Photometric method for determining lactic acid dehydrogenase or glucose-6-phosphate dehydrogenase

ABSTRACT

AN IMPROVED PHOTOMETRIC METHOD FOR THE DETERMINATION OF REDUCED DI- AND TRI-PHOSPHOPYRIDINE NUCLEOTIDES IN AN INCUBATION MIXTURE CONTAINING LACTIC ACID DEHYDROGENASE OR GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND THE APPROPRIATE SUBSTRATES THEREFOR, COMPRISING ADJUSTING THE PH OF THE REACTION MIXTURE TO FORM ABOUT 10.0 TO ABOUT 12.0, PRIOR TO THE PHOTOMETRIC DETERMINATION, TO STABILIZE THE ABSORBANCE OF THE RESULTING SOLUTION WHEN MEASURED AT ABOUT 340 MU.

United States Patent lfice Patented Sept. 24, 1974 U.S. Cl. 195-103.5 R9 Claims ABSTRACT OF THE DISCLGSURE An improved photometric method forthe determination of reduced diand tri-phosphopyridine nucleotides in anincubation mixture containing lactic acid dehydrogenase orglucose-6-phosphate dehydrogenase and the appropriate substratestherefor, comprising adjusting the pH of the reaction mixture to formabout 10.0 to about 12.0, prior to the photometric determination, tostabilize the absorbance of the resulting solution when measured atabout 340 III/.4.

This application is a continuation-in-part of my cpending applicationSer. No. 838,664, filed July 2, 1969, and now abandoned.

This invention relates to an improvement in a direct photometric methodfor the assay of mixtures including the lactic acid dehydrogenase enzymein conjunction with the DPN-DPNH, pyruvate-lactate substrate system orthe glucose 6 phosphate dehydrogenase enzyme in conjunction with theTPN-TPNH, glucose 6 phosphate 6- phosphogluconic acid substrate system.DPN is an abbreviation for diphosphopyridine nucleotide, which is alsoreferred to as nicotinamide adenine dinucleotide, which is alsoabbreviated as NAD. DPNH and NADH are abbreferred to as nicotinamideadenine dinucleotide, which is also referred to as reduceddiphosphopyridine nucleotide. Similarly, TPN is an abbreviation fortriphosphopyridine nucleotide, which is also referred to as nicotinamideadenine dinucleotide phosphate, which is also abbreviated NADP. TPNH andNADPH are abbreviations for the reduced form of this substance, which isalso referred to as reduced triphosphopyridine nucleotide and reducednicotinamide adenine dinucleotidephosphate.

Photometry is generally preferred in enzyme assays over manometric andpH procedures, because of its simplicity, rapidity and usually thecapability of measuring lower enzyme and substrate concentrations.

Enzymes are biochemical catalysts that enable many complex chemicalreactions to take place at ordinary temperatures. An important class ofenzymes are the dehydrogenases, which in the presence of a hydrogenacceptor such as pyridine nucleotide, effect dehydrogenation.

Lactic acid dehydrogenase, which is commonly abbreviated LDH and whichhas the systemic name: L- lactate; NAD oxidoreductase catalyses thereversible oxidation of L-whydroxy monocarboxylic acids. The enzyme isstereospecific for L-lactate and shows high activity with thepyruvate-lactate couple. Thus, LDH catalyses the reaction:

Pyruvate NADH +H+ Lactate NAD+ pyruvate and NADH. When LDH is theunknown, the sample of material thought to contain LDH is incubated inan appropriately buffered mixture with either pyruvate and NADH orlactate and NAD. The terms pyruvate" and lactate refer, respectively, tothe anions and H C-CHOH-COO which are present in the aqueous solution.

Glucose-6-phosphate dehydrogenase, which is commonly abbreviated G6PDHand which has the systemic name: D-glucose 6 phosphate: NADPoxido-reductase catalyzes the reversible oxidation ofglucose-6-phosphate, the product of this oxidation being6-phosphogluconic acid. Glucose 6 phosphate and 6-phosphogluconic acidare commonly abbreviated to G6P and 6-PGA respectively. Thus G6PDHcatalyzes the reaction:

Where an unknown quantity of glucose-6-phosphate is to be determined,appropriate amounts of NADP and G6PDH are incubated in an appropriatelybuffered solution to result in the formation of 6-phosphogluconic acidand in NADPH. When 6-phosphogluconic acid is to be determined,appropriate amounts of NADPH and G6PDH are incubated in thatappropriately buffered solution and the reaction allowed to proceed withthe formation of glucose-6-phosphate and NADP. When G6PDH is theunknown, the sample of material thought to contain GGPDH activity isincubated in an appropriately buffered mixture with either G6P and NADPor 6-PGA and NADPH. The determination of the presence of LDH or G6PDHactivity or of the presence of their respective substrates has been usedfor various purposes in clinical chemistry.

A Wide variety of photometric assays have been used to determine LDHactivity. Colorimetric methods have been based on the formation ofpyruvate 2,4-dinitrophenylhydrazone or upon the formation of a redformazin in colloidal suspension. A method involving an ultravioletspectrophotometer has been used in which the lactate is reacted withB-quinolhydrazine to form a hydrozone having an absorption peak at 305millimicrons (me). A fluorometric method described in US Pat. No.3,384,555 involves the determination of the DPNH by reaction withresazurin in the presence of diaphorase.

The activity of G6PDH canalso be measured by a variety of methods. Inthe presence of nicotinamide and methylene blue the amount of oxygenconsumed in the reoxidation of TPNH to TPN, can be measuredmanometrically. Alternatively, G6PDH activity can be measuredphotometrically by following the rate of reduction of2,6-dichlorophenolindophenol, using phenazine methosulphate [PMS] as anelectron carrier.

LDH and G6PDH activity can be measured spectrophotometrically byfollowing the rate of change of the absorbance at about 340 m theabsorption peak of DPNH and TPNH. DPN and TPN have no absorbance at thisWave length. When using lactate and DPN or G6P and T PN as substrates,the rate of increase of A is measured. When using pyruvate and DPNH or6-PGA and TPNH as substrates, the rate of decrease in A is measured. Theamounts of pyruvate, lactate, G6P or 6-PGA in complex mixtures, can alsobe determined by measuring the changes in the absorbance at 340 Ill/L.

Although 340 m is the normally used wavelength for the determination,higher and lower wavelengths have been used for the determination andwavelength shifters can also be added to increase the efiiciency ofdifferent wavelength. For example, the DPNH can be made to fluoresce bythe addition of suitable fluorescing agents and the fluorescencedetermined using an appropriate filter, e.g. one which transmits the 313and 366 m lines. These equivalent wavelengths will hereafter be referredto as about 340 m This invention relates to an improvement in the latterkinetic approach involving a measure of the rate of change of absorbanceat about 340 m Previously this kinetic approach has not been consideredsuitable for batch analysis as it involves possible error due toinstrument instability, and requires control of the temperature of thecuvette well of the photometer. The paper appearing in Scand. J. Clin.Lab. Invest. 17 265 (1965) proposed to overcome these problems bystopping the reaction of pyruvate to lactate, after a fixed incubationperiod, by the addition of the enzyme inhibitor p-chloromecuribenzoatein highly alkaline solution. However, the stopping of the reaction withan enzyme inhibitor such as p-chloromecuribenzoate is limited by thetoxicity of the compound to humans, the poor stability of its solutions,the danger of residues being left on glassware which inhibit futureenzyme assays, and the poor stability of the absorbance of the stoppedenzyme action.

In the procedures of this invention, both the forward and the reversereactions of the enzymes can be stopped and the resulting absorbancestabilized by adjusting the pH of the reaction mixture after incubationto from about 10.0 to about 12.0, and preferably to about 10.5.

More particularly, this invention relates to the improvement in thespectrophotometric method for the determination of DPNH or TPNH in thepresence of LDH or G6PDH respectively. Thus, after a suitable incubationperiod for reaction, the pH of the reaction mixture is adjusted to fromabout 10.0 to about 12.0, and preferably to about 10.5, to stabilize theresulting absorbance of the solution, without using elfective amounts ofan enzyme poison.

The term photometric refers to the use of an instrument measuringrelative radiant power as a function of wavelength, using either afilter, prism or grating. Photometers which disperse light by prism orgrating into a spectrum from which the desired band is isolated bymechanical slits are termed spectrophotometers. Spectrophotometers areusually more sensitive than filter photometers. The terms LDH, pyruvate,lactate, DPN, DPNH, G6PDH, G61, 6-PGA, TPN, and TPNH are defined asbefore. The term buffered solution refers to an aqueous solutioncontaining the appropriate buffers which are used or known to besuitable for use in the enzyme reaction and subsequentspectrophotometric reading at 340 mg. The most common buffered solutionsfor use in the enzyme reaction in question are phosphate andpyrophosphate, although other buffers have been used in the reaction.The buffer of choice for the enzyme reaction is one for which thenegative log of the apparent dissociation constant (abbreviated pK is ator near the optimum pH of the enzyme in question.

The pH of the reaction mixture can be adjusted to the range of fromabout 10.0 to 12.0 to stop the enzyme reaction and to stabilizeabsorbance at about 340 Ill .t by the addition of a suitable hydroxylion (OH containing base. To minimize problems of the formation ofspectral- 1y interfering substances, it is preferred to use an inorganichydroxide base, preferably an alkali metal hydroxide such as sodium orpotassium hydroxide. The volume of base added to the reaction mixture isnot critical so long as the desired pH is reached. However readingerrors on the spectrophotometer are minimized when the percenttransmittance is between 20 and 70 percent. Thus it is desirable to usea base of an appropriate hydroxide concentration in an appropriateamount to afford the designated percent transmittance when used with therange of DPNH to be detected.

Because the enzyme reaction mixture is incubated in a buffered solutionwith a buffer that has a pK lower than 10.0 to 12.0, the pH of thesolution after the addition of 4 base is often lower than the pH of theadded base. To avoid the problem of having to determine the pH of boththe base added and the pH of the final solution, it is desirable to addthe base in the form of a buffered solution.

The buffered bases desirable for use in altering the pH of the reactionmixture are those having a pK of 9 or greater. Thus examples of thepreferred buffered bases for use in adjusting the pH are borate andcarbonate buffers which in a 0.1 M concentration have pK is of,respectively, about 9.2 and 9.9. Thus, the addition of ten volumes of0.1 M borate and carbonate buffers, pH 10.5 to one volume of 0.1 Mphosphate buffer containing the enzyme reaction mixture and having a pHof about 7 can result in a stopped enzyme reaction and stabilizedmixture with a pH above about 10.0. On the other hand the use of bulferswith low pK s, e.g. use of 0.1 ,M pH 10.5 Tris, pyrophosphate, sodiumbarbital, glycylglycine or triethanolamine buffer in the same proportion to the orthophosphate bulfered solution can result in pHssubstantially lower than about 10.0 and a failure to stabilize theabsorbance of the mixture at about 340 mg.

The pH to which the enzyme mixture is adjusted to stabilize theabsorbance at 340 m is critical. The critical range for pH adjustment isabout 10.0 to about 12.0. The term about is used to indicate that the pHadjustment can be made to a point a few tenths of a pH point below 10.0or above 12.0 without unacceptably alterating the stability of thesystem. However acceptable stability is not achieved if the pH isadjusted to a point lower than 9.7 or higher than 12.3. Moreover, whenusing buffered base, the preferred range for stability is about 10.0 toabout 11.0. The preferred pH range for adjustment is that which gives noobservable absorbance change over a three hour period at temperatures upto the denaturation point of the enzyme involved. Nevertheless, anabsorbance change over the three hour period of up to 7% with certainspectrophotometers may be considered acceptable for certain work.

The improvement for the determination of DPNH of this invention can beused in assays where lactic acid dehydrogenase is the unknown, in whichcase the sample thought to contain LDH is incubated with a bufferedsolution containing either pyruvate and DPNH (in which case thedetermination is made using the forward reaction) or containing lactateand DPN (in which case the reverse reaction is used). Likewise, theimprovement of this invention can be used in assays for lactate orpyruvate, in which case known amounts of LDH and either DPNH or DPN areadded to the reaction mixture. Similarly, the improvement of thisinvention can be used in an assay for DPNH or DPN, in which case knownamounts of LDH and pyruvate or lactate are added to the reactionmixture. In all these cases, however, it is the DPNH or change inconcentration thereof, that is the basis for the determination.

In a similar manner the improvement in the determination of the TPNH canbe used in assays where any one of glucose-6-phosphate dehydrogenase,G6P, TPN, 6-PGA, or TPNH is the unknown. In all these cases, however, itis the TPNH or change in the concentration thereof, that is the basisfor the determination.

In determining unkown lactate or pyruvate, the LDH enzyme system can beused as an indicator. For example it can be used in determining theactivity of glutamicpyruvic acid transaminase (GPT). The latter enzymecatalyzes the following reaction:

L-alanine wketoglutarate glutamate pyruvate The activity of an unkownamount of GPT can thus be determined by adding to the above reactionmixture approprrate amounts of DPNH and LDH, then determining the rateof change of absorbance of the DPNH at about 340 m and using theimprovement of this invention to stop the LDH enzyme reaction andstabilize the absorption at the wavelength. Likewise, in thedetermination of creatine phosphokinase (CPK) which catalyzes thefollowing reaction:

CPK Creatine ATP ereatine phosphate ADP There can be added to the systemphosphoenolpyruvate and (PK) to result in the reaction:

ADP phosphoenolpyruvate ATP pyruvato Thus the rate of change inabsorbance at 340 m upon the addition of appropriate amounts of DPNH andLDH to this system serves as an indicator of the CPK activity; and theimprovement of this invention can be used to stop the LDH reaction andto stabilize the resulting absorbance.

The G6PDH enzyme system can also be used as an indicator when coupled toother enzyme systems. Following the reaction of these complex systems,the improvement of the present invention can be used to stabilize theabsorbance of the TPNH when measured at about 340' m For example, theG6PDH enzyme system can be used in the determination of CPK activity. Inthis case the reverse reaction of CPK to that shown above, is utilized,in which creatine phosphate and ADP react with the formation of creatineand ATP. There can then be added to the system, glucose and hexokinasewhich results in the reaction below:

The G6P thus formed can then be utilized in the G6PDH reaction systemand the improvement of the present invention can be used to stabilizethe absorbance of TPNH when measured at about 340 m,u..

The methods for all these determinations, with the exception of thefinal pH adjustment of this invention, are described and known in theprior art and can be used in the procedure of this invention.

The following examples are given to further illustrate the invention.Further modifications will also occur to those skilled in the art andthus the examples should not be considered limiting in spirit or inscope. In these examples temperatures are given in degrees centigradeC.), amounts of materials in grams (gms.), milligrams (mgs.), moles (M)or millimoles (mM.), liters (1.) and milliliters (mL), concentrations ofsolutions as, molar (Molar) and millimolar (mMolar), wavelengths inmillimicrons (my), and lengths in millimeters (mm).

EXAMPLE 1 Preparation of Reagents Pyruvate Substrate.A 0.1 Morthophosphate buffer, pH 6.75 at 37 is prepared by mixing appropriateamounts of disodium hydrogen orthophosphate and sodium dihydrogenphosphate in distilled water to afford a 0.1 Molar solution of pH 6.75at 37. The 1.59 mM. pyruvate substrate is prepared by adding sufiicientsodium pyruvate to that 0.1 M orthophosphate butter to afford a solutioncontaining 1.59 millimoles of sodium pyruvate per liter oforthophosphate bufi'er. This solution can be preserved by adding a smallamount of chloroform as a preservative. The chloroform-stabilizedsolution is stable for over a year if refrigerated at 2 tofl-DPNH-Pyruvate Working Solution.Ten mg. of reduced[i-diphosphopyridine nucleotide (B-DPNH) is mixed with 10 ml. of thepyruvate substrate prepared as above. The solution is stable for atleast 2 hours at room temperature and at least 6 hours if refrigerated.

Lactic Acid Dehydrogenase (LDH).-The preparation used is a control serumwhich contains lactic acid dehydrogenase (LDH) obtained from beef heart(sold by General Diagnositics, a Versatol-E or Versatol-EN) having adesignated activity in either International Units per ml. or inWroblewski-La Due units per ml. One In ternational Unit of Activity isdefined as that amount of enzyme which causes an initial rate ofoxidation of 1 micromole (,amole) of DPNH per minute per liter at 25under the conditions specified by the International Union ofBiochemistry.

One Wroblewski-La Due unit is defined as that amount of enzyme whichcauses a decrease in the absorbance of DPNH at 340 m of 0.001/min./ml.of samples at 32 under the conditions specified in Proc. Soc. Exp. Biol.Med. 210 (1955).

Procedure: The procedure used indicates the ability of the various testsolutions used to stop the enzyme reaction. For each test solution used,0.5 ml. of the ,8-DPNH-pyruvate working solution is placed into each oftwo, 16.8 mm. internal diameter, 19 mm. external diameter round cuvetteswhich have been matched at 340 m for this assay. The cuvettes are thenplaced in a water bath held at 37 and allowed to stand there for about 2to 3 minutes. Then 0.05 ml. of LDH serum is added to one of the cuvetteswith mixing. The LDH serum used here (Versatol-E Lot 0118038) contains370 International Units per liter, which is equivalent to 768Wroblewski-La Due per ml. Incubation of both cuvettes is continued at 37for exactly 5 minutes. At the end of that five minute period 5.0 ml. ofthe test solution is added to each of the two cuvettes with mixing. Then0.05 ml. of the LDH serum is added with mixing to the cuvettes in whichno serum had been placed during incubation; this cuvette serves as thecontrol sample. The cuvette which has been incubated for 5 minutes withthe enzyme serves as the test sample. The percent transmission for boththe cuvettes is read on a Coleman Junior II 6/ 20 spectrophotometer at340 m which has been previously calibrated for accuracy at thatwavelength. The first reading, i.e. 0 time, is taken immediately afterthe buffer addition in the case of the test sample and the bufferaddition followed by the serum addition in the case of the controlsample. Subsequent readings are taken 10, 20, 30, 40, 45, 60, andminutes after 0 time. Between readings the samples stand at roomtemperature of about 27. For a given reading time the difference inabsorbance of the control sample versus the test sample (AA/5 min.) isobserved from the instrument or can be calculated from the formula:

Absorbance=2log percent transmission =log (100/ percent transmission(AA/5 min. at TAA/5 min. at 0) 100 AA/5 min. at 0 =percent changeVarious concentrations of sodium hydroxide in distilled water were usedas test samples. The normality of representative test solutions used aregiven in the first column of Table 1 below. The pH of the test andcontrol samples atfer the particular NaOH addition is given in thesecond column. The third column gives the AA/S min. for each of thetimed readings and the percent change of this AA/ 5 min. from the 0 timereading.

In the samples having a final pH of 7.9, the absorbance of both thecontrol and test samples and the individual values for absorbance (A)decreased over the time interval studied. The percent change in thesamples having a pH of 9.46 and 9.84 represent decreases in absorbancefor both the control and test samples over the period studied. In thesamples of pH 12.10, the absorbancy of the control sample remainssubstantially constant throughout the time interval, while theabsorbancy of the test sample increased. The percent change for thesamples of pH 12.34 and 13.2 represent ultimate decreases in theabsorbancy for both the test and control samples. The negativedifference in absorbancy for the samples of pH 13.8 show that the highconcentration of base has completely altered the system.

8 of Versatol-E, Lot 0087027, which contains 461 International Units perliter, which is equivalent to 955 Wroblewski-La Due units per ml.,rather than the lot used in Example 1.

TABLE 1 pH of sample after Time at Na() 11' Normality NaOH solutionadded addition 00 1'20 130 (a) 0.00117 7. 00 Ali/5 11111111105. .112 007080 072 050 018 017 0. 12 Percent change 13 20 36 -57 80 (b) 0. 00625 9.46 AA/5 minutes. .136 136 136 .136 1.36 140 137 127 Percent change 0 0 00 +3 +1 -7 (e) 0.00714 9. 84 AA/S minutes. 114 .114 .114 114 .114 114.113 .113 Percent change- 0 0 0 0 0 +1 1 ((1) 0.00833 10. 26 AA/Sminutes. .120 120 120 120 120 120 120 Percent change 0 0 0 0 0 0 0 (e)0.0100 10.78 AA/S minutes... .113 .113 113 113 113 113 113 113 Percentchange 0 0 0 0 0 0 0 (t) 0.025 (pH 11.8) 12.0 AA/5 minutes. 143 143 143143 143 143 .143 143 Percent change 0 0 0 0 0 0 0 (g) 0.0312 12. 10 AA/Sminutes. .098 006 0.94 080 .086 086 086 086 Percent change 2 4 9 12 -1212 12 (11) 0.0417 12. 20 AA/E minutes. 000 .090 090 000 000 000 090 000Percent change 0 0 0 O 0 0 0 (i) 0.125 12.34 AA/5 minutes. 104 .104 104.102 .106 108 107 110 Percent change 0 0 2 +2 +4 +3 +6 (j) 0.25 (pH12.0) 13.2 AA/5 minutes. .125 .105 118 .132 .134 1140 .143 Percentchange l6 6 +6 +7 +12 +10 +11 (k) 2.5 N (pH 13.4) 13. 8 1111/5 minutes.070 054 0 024 010 010 010 .010

EXAMPLE 2 EXAMPLE 3 0.1 M borate bufiers of varying pH are prepared bydissolving 6.184 grams of boric acid in suflicient carbonate-free,aqueous sodium hydroxide to afford a solution of the desired pH uponadding sufiicient distilled water to make a final volume of one liter.The final pH is checked and adjusted with 0.1 N sodium hydroxide ifnecessary. For example to prepare a 0.1 M borate buffer, pH 10.0, 6.184grams of boric acid are dissolved in 439 ml. of 0.1 N aqueous sodiumhydroxide; the pH is checked and adjusted as necessary with 0.1 N sodiumhydroxide.

Substitution of 5 ml. of 0.1 M borate buffer, pH 10.5 for the sodiumhydroxide in the procedure of Example 1, results in a mixture having afinal pH of about 10.1. The absorbance of both the control sample andthe test sample was unchanged throughout the minute reading period, thusresulting in no change in the AA/S min. readings for the individualsamples. Identical stability is observed when the samples are held at 37throughout the 180 minute reading period.

The substitution of 5 ml. of 0.1 M borate buffers, having pHs of,respectively, 9.0, 9.2, 9.4, 9.6 and 9.8 for the 5 ml. of aqueous sodiumhydroxide in the procedure of Example 1, all resulted in decreases inthe AA/S min. over the 180 minute reading period. These decreases Weredue to decreases in absorbancy for both the control and test samples.Thus using 0.1 M borate butter, pH 9.0 the AA/S min. at 0 time was0.304, but by the time of the 180 minute reading was 0.213, a percentdecrease in AA/5 min. of 29%. For the 0.1 M borate buffer, pH 9.8 theAA/S min. at 0 time was .271, while the figure at the 180 minute readingwas .264, a percent decrease of 4%.

The substitution of 5 ml. of 0.1 M borate buffers, having pHs of,respectively, 10.0, 10.2, 10.4, 10.6, 10.8 and 11.0 resulted in sampleswith no significant change in absorbance over the 180 minute readingperiod. 0.1 M borate bufl'ers with higher pHs, up to 11.8 can be used,but result in percent changes in absorbancy over the 180 minute readingperiod varying from 0% to 4%.

The LDH serum used in this example was 0.05 ml.

0.1 M carbonate buffers are prepared by mixing appropriate amounts ofdisodium carbonate and sodium hydrogen carbonate with distilled water togive the desired pH. For example 0.1 M carbonate buffer, pH 10.0 at 25is prepared by mixing 58 ml. of 0.1 M aqueous disodium carbonatesolution with 42 m1. of 0.1 M aqueous sodium hydrogen carbonatesolution.

Substitution of 5 ml. of 0.1 M carbonate buffer, pH 10.0 in theprocedure of Example 1 for the 5 m1. of aqueous sodium hydroxide,results in stabilization of the absorbance of the test and controlsamples over the 180 minute reading period. Use of 0.1 M carbonatebutter pH 9.0 fails to stabilize the absorbance, resulting in a percentdecrease in AA/ 5 min. at the 180 minute reading of roughly 20%. Use of0.1 M carbonate buffer, pH 11.9 resulted in a percent in AA/S min. atthe 180 minute reading of 0%, but with an increase in the AA/S min. atthe 10, 20, 30 and 40 minute readings.

EXAMPLE 4 0.1 M 2-amino-2-methyl-l-propanol buffer, pH 10.5 is preparedby adding appropriate amounts of 2-amino-2- methyl-l-propanol todistilled water then adjusting the pH to 10.5 with hydrochloric acid.Substitution of 5 ml. of 0.1 M 2-amino2-rnethyl-l-propanol buffer, pH10.5 for the 5 ml. of aqueous sodium hydroxide in the procedure ofExample 1, results in a final pH for the mixture of 10.32. Theabsorbance of the control and test samples are stabilized over the 180minute reading period, the percent change in AA/5 min. over that periodbeing equal or less than 2%.

The use of equivalent amounts of 0.1 M, pH 10.5; tris, pyrophosphate,sodium barbital, glycylglycine and triethanolamine buffers,respectively, in the procedure of Example 1 results in final pHs of 9.4or less and decreases in absorbance for both the test and controlsamples over the 180 minute reading. The percent decrease in AA/5 min.at the 180 minute reading ranges from 14% in the case of theglycylglycine buffer having a final pH of 9.38 to 19% in the case of thesodium barbital bulTer, where the pH of the resulting samples is 9.22.

9 EXAMPLE 5 A buffered aqueous substrateis prepared consisting ofdistilled water containing sodium pyrophosphate, L(+) lactic acid anddiphosphopyridine nucleotide (DPN) in a concentration per liter of,respectively, 0.05 M, 77.5 mM. and 5.25 mM., respectively, and having apH of 8.80. 0.5 ml. of this buifered substrate is placed into each offour round cuvettes, which have an external diameter of 19 mm. and aninternal diameter of 16.8 mm. and which have been previously matched at340 mu wavelength. The cuvettes are placed in a water bath held at 37for about 2 minutes to bring the contents up to this temperature. Intoeach of three of the four cuvettes there is added with mixing 0.05 ml.of control serum (Versatol-E, Lot 0087027, which contains 185International Units of lactic acid dehydrogenase per liter, which isequivalent to 382 Wacker Units per ml. Incubation is continued at 37 inthe first tube to which enzyme has been added for 2 minutes, in thesecond tube for 5 minutes and in the third tube for 10 minutes. At theend of these time intervals 5.0 ml, of 0.1 M borate buffer, pH 10.0prepared as in Example 2 is added to the tubes with mixing. To the tubethat originally received no enzyme, there is added 5.0 ml. of the 0.1 Mborate buffer and 0.05 ml. of the control serum; this cuvette serves asthe control sample. The 2, 5 and 10 minute incubation mixtures serve asthe test samples. As in Example 1, readings of percent transmission aremade on a Coleman Junior II 6/20 spectrophotometer at 340 m which hasbeen previously calculated for accuracy at that wavelength. The firstreading, i.e. time is taken immediately after the buffer addition in thecase of test samples and the buffer addition followed by the serumaddition in the case of the control sample. Subsequent readings aretaken at 10, 20, 30, 40, 45, 60, 120 and 180 minutes after the 0 time.

The use of 0.1 M borate bulfer, pH 10.0 in the above procedure resultsin absorbance uniformity for each of the control and test samplesthroughout the 180 minute reading period.

EXAMPLE 6 The procedure of Example 1 is repeated except that in thefirst series of cuvettes ml. of 0.0555 N sodium hydroxide containing0.555% by weight p-chloromecuribenzoate is used in place of the 5 m1. ofsodium hydroxide used in that Example 1 and in the second series oftubes 5 ml. of 0.0555 N sodium hydroxide is used in place of the 5 ml.of sodium hydroxide in the concentrations used in Example 1.

In the cuvettes containing the p-chloromecuribenzoate in sodiumhydroxide, the final pH was 12.02, the absorbance for both the controland the test sample increased during the 180 minute reading period, theAA/S min. at 0 time being .075 and at the 180 minute reading being .089,a percent increase of AA/S min. of 18.7 percent. On the other hand usingthat concentration and amount of sodium hydroxide without thep-chloromecuribenzoate afforded a final pH of 12.02, the AA/S min. at 0time being .092 and at the 180 minute reading being .091, a change ofonly 1%. Thus it appears that better absorbance stability at theindicated pH is achieved without the use of the enzyme poisonp-chloromercuribenzoate.

EXAMPLE 7 The procedure of Example 1 is carried out except that 5.0 ml.of 0.1 M borate buffer, pH 10.5 (prepared as in Example 2) issubstituted for the 5.0 ml. of the sodium hydroxide solution used inthat Example 1, and 0.05 ml. of human serum obtained from, respectively,apparently healthy humans, autopsy-proven cases of myocardial infractionand autopsy-proven cases of infectious hepatitis, is substituted for the0.05 ml. of LDH control serum used in Example 1. The absorbance of theresulting solution was unchanged throughout the 180 minute readingperiod.

Although the pH of the incubation mixture used in Example 1 and in thepreceding paragraph is 6.75, lower and higher pHs of the incubationmixture can be used depending on the pH optimum of the LDH enzymes underconsideration. Thus, although the pH optimum for the LDH enzymes foundin apparently-healthy humans is about 65:02, the isoenzymes present incertain disease states may have higher or broader, or both, optimum pH.Although pH of 6.75 used in the described procedure minimized variationsdue to the different pH optimum of the isoenzymes present, satisfactoryresults can be obtained by carrying out the incubation at a pH as highas 7.0 or in some cases as high as 7.5 and 8.0.

EXAMPLE 8 The G6PHD enzyme system can be used as an indicator in themeasurement of CPK activity and following the reaction catalyzed by CPKthe improvement of the present invention can be used to stabilize theabsorbance of the TPNH so formed.

Preparation of Reagents The following substrates, enzymes and cofactorswere dissolved in distilled water to give the final concentrationsindicated: creatine phosphate 1.7 mM.; TPN, 4 mM., ADP, 2 mM.;hexokinase, 1 Unit/ 3 mls.; G6PDH, 1 Unit/ 3 mls.; glucose 10 mM.;MgSO;;, 5 mM.; cysteine, 4 mM.; tris buffer, 0.07 M, pH 7.0; AMP 20 mM.The reagent prepared in this form was stable for at least -6 hours atroom temperature or 2 days when refrigerated,

The stabilizing solution was prepared by dissolving 30.6 grams potassiumtetraborate tetrahydrate in distilled water, bringing this solution topH 10.5 with sodium hydroxide and diluting the whole solution to 1liter.

Procedure: The G6PDH enzyme system in conjunction with the Hexokinasesystem and substrates for the CPK reaction can be used to measure theCPK activity of serum. Thus, tubes containing 1.0 mls. of the complexsubstrate enzyme cofactor solution, as described above, are warmed at 37centigrade in a water bath for several minutes. Then, 50 ,ul. of serumis added to the relevant tubes and after 30 minutes incubation, 5.0 mls.of stabilizer solution is pipetted into each tube. The absorbance of thesolution is then measured at 340 m The CPK activity is then obtainedfrom the equation below:

International Units (IU) :W [Calibration Factor] where:

340 sample' blank D=20,000 [converts 50 ,ul. of serum to 1 liter] V=l.05ml. [volume of reactants] S=5.76 [compensates for dilution bystabilizer] F=0.58 [converts CPK activity to IU at 30 C.]

t=30 rnin. [duration of incubation] e=6.22 cm. ,umole [extinctioncoefficient of NADPH] d=l cm. [length of light path] Results: The CPKactivity of 2 sera were assayed by the procedure above. After theaddition of the stabilizing solution, the absorbance was measured every60 minutes for 5 hours. In both cases the increase in absorbance wasslight during this time, amounting to less than a 2% rise at the end of5 hours. This example demonstrates the ability of the stabilizingreagent to prevent further generation of TPNH in the above enzymesystem.

EXAMPLE 9 The improvements of the present invention can be used in'thedetermination of the pH optimum of the coupled CPK and G6PDH enzymereactions.

The complex enzyme/substrate/cofactor reagent was prepared in anessentially similar manner to that described above. However, prior tothe addition of the serum sample the pH of the substrate/enzyme solutionwas adjusted 11 to one of four values between 6.6 and 7.2. The CPKactivity of the serum samples was then determined at the four differentpH values and the activity was calculated as described before.

The CPK activity of the serum was similar at each of the four pH valuestested, although optimum pH was approximately 6.8.

What is claimed is:

1. In the method for determination of lactic acid dehydrogenase orglucose-6-phosphate dehydrogenase activity wherein the enzymaticreduction or oxidation of di-or triphosphopyridine nucleotide coenzymesin the presence of lactic acid dehydrogenase or glucose-6-phosphatedehydrogenase and its substrate is measured by the rate of change in thespectrophotometric absorbance of said coenzymes, the improvementcomprising terminating the enzymatic reaction by adjusting the pH of thereaction to -12 by means of the addition of approximately 10 volumes ofa buffer solution selected from the group consisting of aqueous alkalimetal hydroxide, aqueous borate and aqueous carbonate.

2. The process of Claim 1 in which the substrate-coenzyme combinationfor lactic acid dehydrogenase is either pyruvate and reduceddi-phosphopyridine nucleotide or lactate and di-phosphopyridinenucleotide.

3. The process of Claim 1 in which a substrate-coenzyme combination forglucose-6-phosphate dehydrogenase is either glucose-6-phosphate andtri-phosphopyridine nucleotide or 6-phosphogluconic acid and reducedtriphosphopyridine nucleotide.

4. The process of Claim 2 in which the pyruvate is generated as a resultof the reaction of creatine and adenosine triphosphate, in the presenceof creatine phosphokinase followed by the reaction of the adenosinediphosphate so formed with phosphenol pyruvate in the presence ofpyruvatekinase.

5. The process of Claim 3 in which the glucose-6-phosphate is generatedas a result of the reaction of creatine phosphate and adenosinediphosphate in the presence of creatine phosphokinase followed by thereaction of the adenosine triphosphate with glucose in the presence ofhexokinase.

6. The process of Claim 1 in which the pH is adjusted to about 10.5.

7. The process of Claim 6 in which the buffer solution is an aqueousalkali metal hydroxide.

8. The process of Claim 6 in which the buffer solution is aqueous sodiumhydroxide.

9. The process of Claim 1 in which the pH of the reaction mixture isadjusted to a pH in the range of from about 10.0 to about 11.0.

References Cited UNITED STATES PATENTS 7/1967 Struck et a1 -103.5 R

OTHER REFERENCES ALVIN E. TANENHOLTZ, Primary Examiner

